Environmental Stress-Inducible 996 Promoter Isolated from Rice and Uses Thereof

ABSTRACT

The present invention relates to environmental stress-inducible 996 promoter isolated from rice, a recombinant plant expression vector comprising said promoter, a method of producing a target protein by using said recombinant plant expression vector, a method of producing a transgenic plant using said recombinant plant expression vector, a transgenic plant produced by said method, a method of improving resistance of a plant to environmental stress by using said promoter, and a primer set for amplification of said promoter.

CLAIM OF PRIORITY

This application makes reference to, incorporates the same herein, andclaims all benefits accruing under 35 U.S.C. §119 from an applicationearlier filed in the Korean Intellectual Property Office on 16 Apr. 2009and there duly assigned Serial No. 10-2009-0033015. This applicationalso makes reference to, incorporates the same herein, and claims allbenefits accruing under 35 U.S.C. §365(c) of my PCT Internationalapplication entitled ENVIRONMENTAL STRESS-INDUCIBLE 996 PROMOTERISOLATED FROM RICE AND USES THEREOF filed on 16 Sep. 2009 and dulyassigned Serial No. PCT/KR2009/005257.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to environmental stress-inducible 996promoter isolated from rice and use thereof. Specifically, the presentinvention relates to environmental stress-inducible 996 promoterisolated from rice, a recombinant plant expression vector comprisingsaid promoter, a method of producing a target protein by using saidrecombinant plant expression vector, a method of producing a transgenicplant using said recombinant plant expression vector, a transgenic plantproduced by said method, a method of improving resistance of a plant toenvironmental stress by using said promoter, and a primer set foramplification of said promoter.

2. Description of the Related Art

A promoter is a part of genome which is located upstream of a structuralgene and regulates transcription of the structural gene into mRNA. Apromoter is activated by binding of various general transcriptionfactors, and it typically comprises a base sequence such as TATA box,CAT box, etc. which regulates gene expression. Since proteins that arerequired for basic metabolism of a living body need to be maintained atconstant concentration, a promoter that is associated with genes of suchproteins is constantly activated even by general transcription factorsonly. On the other hand, for the proteins of which function is notrequired during normal time but required only under specificcircumstances, an inducible promoter which can induce expression of acorresponding structural gene is linked to the corresponding genes. Inother words, an inducible promoter is activated by binding of specifictranscription factors that are stimulated during a developmental processof an organism or stimulated by environmental factors.

For the development of a transgenic plant based on gene transformation,a promoter which can induce constant and strong expression, for examplecauliflower mosaic virus 35S promoter (CaMV35S, Odell et al., Nature313: 810-812, 1985), is widely used. However, since constantoverexpression of a specific gene linked to such promoter can produce anexcess amount of proteins that are not necessarily required formetabolism of a normal living body, it often becomes problematic in thata transgenic plant does not properly germinate or only a small-sizedplant is produced due to toxic effect generated by the proteins presentin an excess amount. As a representative example, it was found thatArabidopsis thaliana in which DREB1A, a gene encoding transcriptionfactors responding to environmental stress, is overexpressed by usingCaMV35S promoter has improved resistance to low temperature and droughtcondition, but also had a dwarfed phenotype with inhibited growth andincreased production of proline and water-soluble carbohydrates (Liu etal., Plant Cell 10: 1391-1406, 1998; Gilmour et al., Plant Physiol. 124:1854-1865, 2000). Occurrence of such problems can be minimized by usingan inducible promoter for rd29A, that is a gene relating toenvironmental stress, instead of CaMV35S promoter (Kasuga et al, Nat.Biotechnol. 17: 287-291, 1999).

Under the circumstances, instead of a promoter which constantly inducesgene expression in a plant tissue, an inducible promoter which caninduce expression of a target gene at specific time and under specificcondition has been actively studied and developed. As a scientificresearch, an inducible promoter system, which can stimulate biosynthesisof a target protein based on introduction of a promoter isolated from amicroorganism or an animal to a plant and use of a chemical substance asan inducing agent, has been widely developed. Until now, as an exampleof application of an expression inducing system based on use of achemical substance on a plant, a method in which a steroid such asdexamethasone, antibiotics such as tetracycline, and a chemicalsubstance such as copper ion, IPTG and the like are used as an inducingagent has been known (Gatz et al., Plant J. 2: 397-404, 1992; Weimann etal., Plant J. 5: 559-569, 1994; Aoyama T. and Chua N-H, Plant J. 11:605-612, 1997). However, such method is problematic in that the chemicalsubstance used as an inducing agent for a system is extremely expensiveand such chemicals often have a toxic effect by themselves, so that themethod cannot be applied to all plants.

In Korean Patent Registration No. 10-0781059, an inducible promoter thatis activated by environmental stress and a method of obtaining atransgenic protein in guard cells by using such promoter are described.In addition, in Korean Patent Registration No. 10-0578461, astress-inducible promoter that is isolated from rice is disclosed.However, these are all different from the promoter described in thepresent invention.

The present invention was devised in view of above-described needs.Specifically, after intensive studies to develop a promoter which isinduced by environmental stress, particularly by drought stress,inventors of the present invention found that a certain promoterisolated from rice is strongly induced by drought stress, and thereforecompleted the present invention.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention providesenvironmental stress-inducible 996 promoter isolated from rice.

Further, the present invention provides a recombinant plant expressionvector comprising said promoter.

Further, the present invention provides a method of producing a targetprotein by using said recombinant plant expression vector.

Further, the present invention provides a method of producing atransgenic plant using said recombinant plant expression vector.

Further, the present invention provides a transgenic plant produced bysaid method.

Further, the present invention provides a method of improving resistanceof a plant to environmental stress by using said promoter,

Still further, the present invention provides a primer set foramplification of said promoter.

The present invention provides an isolated nucleic acid constructcomprising a strong constitutive plant promoter which can bind to aheterologous gene encoding a desired polypeptide in a tissue of atransgenic plant. When used as a construct for a heterologous codingsequence in a chimeric gene, said construct functions as a droughtstress inducible-promoter and is useful for obtaining high expression ofa desired polypeptide in a leaf tissue of a plant. A vector in which acertain chimeric gene is linked to said construct can be introduced to aplant tissue, and as a result, a plant can be deliberately transformedwith such vector and a foreign material can be produced from theresulting transgenic plant.

In addition, it is expected that, by using the promoter that is providedby the present invention and a technology of breeding an environmentalstress-resistant transgenic plant using the promoter, improvement ofcrop yield for the economically important crops can be achieved.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention, and many of the attendantadvantages thereof, will be readily apparent as the same becomes betterunderstood by reference to the following detailed description whenconsidered in conjunction with the accompanying drawings, in which likereference symbols indicate the same or similar components, wherein:

FIG. 1 is a schematic diagram showing pGEMTvec996 promoter for droughtstress-inducible 996 promoter isolated from rice.

FIG. 2 is a schematic diagram showing pHC10::996 promoter vector fortransformation delivery of drought stress-inducible 996 promoterisolated from rice.

FIG. 3 is a RT-PCT result showing the expression of droughtstress-inducible 996 promoter gene isolated from rice.

FIG. 4 is a GFP assay result for determination of the expression ofdrought stress-inducible 996 promoter gene isolated from rice. 996p-1,-2, -3, -4, -5, -6, -7, -8, -9 and -10 each represent anindependently-transformed plant.

DETAILED DESCRIPTION OF THE INVENTION Mode for the Invention

In order to achieve the object of the invention described above, thepresent invention provides environmental stress-inducible 996 promoterisolated from rice, which consists of a nucleotide sequence of SEQ IDNO: 1.

The present invention is related to a specific promoter that is isolatedfrom rice, and more specifically, the promoter consists of a nucleotidesequence of SEQ ID NO: 1. Further, variants of the said promotersequence are within the scope of the present invention. The variantshave a different nucleotide sequence but have similar functionalcharacteristics to those of the nucleotide sequence of SEQ ID NO: 1.Specifically, the promoter of the present invention may comprise anucleotide sequence with at least 70%, preferably at least 80%, morepreferably at least 90%, and most preferably at least 95% homology withthe nucleotide sequence of SEQ ID NO: 1.

The “sequence homology %” for a certain polynucleotide is identified bycomparing a comparative region with two sequences that are optimallyaligned. In this regard, a part of the polynucleotide in comparativeregion may comprise an addition or a deletion (i.e., a gap) compared toa reference sequence (without any addition or deletion) relative to theoptimized alignment of the two sequences.

With respect to the promoter according to one embodiment of the presentinvention, the environmental stress can be drought, high temperature,cold temperature, high salt, heavy metals, etc. Preferably, it isdrought stress.

In order to achieve another object of the present invention, the presentinvention provides a recombinant plant expression vector which comprisesthe promoter according to the present invention. As an example of therecombinant plant expression vector, pHC10::996 promoter vector shown inFIG. 2 of the present invention can be mentioned, but not limitedthereto.

The term “recombinant” indicates a cell which replicates a heterogeneousnucleotide or expresses the nucleotide, a peptide, a heterogeneouspeptide, or a protein encoded by a heterogeneous nucleotide. Recombinantcell can express a gene or a gene fragment, that are not found innatural state of cell, in a form of a sense or antisense. In addition, arecombinant cell can express a gene that is found in natural state,provided that said gene is modified and re-introduced into the cell byan artificial means.

The term “vector” is used herein to refer DNA fragment (s) andnucleotide molecules that are delivered to a cell. Vector can be usedfor the replication of DNA and be independently reproduced in a hostcell. The terms “delivery system” and “vector” are often interchangeablyused. The term “expression vector” means a recombinant DNA moleculecomprising a desired coding sequence and other appropriate nucleotidesequences that are essential for the expression of theoperatively-linked coding sequence in a specific host organism.Promoter, enhancer, termination codon and polyadenylation signal thatcan be used for an eukaryotic cell are well known in the pertinent art.

A preferred example of plant expression vector is Ti-plasmid vectorwhich can transfer a part of itself, i.e., so called T-region, to aplant cell when the vector is present in an appropriate host such asAgrobacterium tumefaciens. Other types of Ti-plasmid vector (see, EP 0116 718 B1) are currently used for transferring a hybrid gene toprotoplasts that can produce a new plant by appropriately inserting aplant cell or hybrid DNA to a genome of a plant. Especially preferredform of Ti-plasmid vector is a so called binary vector which has beendisclosed in EP 0 120 516 B1 and U.S. Pat. No. 4,940,838. Other vectorthat can be used for introducing the DNA of the present invention to ahost plant can be selected from a double-stranded plant virus (e.g.,CaMV), a single-stranded plant virus, and a viral vector which can beoriginated from Gemini virus, etc., for example a non-complete plantviral vector. Use of said vector can be advantageous especially when aplant host cannot be appropriately transformed.

Expression vector would comprise at least one selective marker. Saidselective marker is a nucleotide sequence having a property based onthat it can be selected by a common chemical method. Every gene whichcan be used for the differentiation of transformed cells fromnon-transformed cell can be a selective marker. Example includes, a generesistant to herbicide such as glyphosate and phosphintricin, and a generesistant to antibiotics such as kanamycin, G418, bleomycin, hygromycin,and chloramphenicol, but not limited thereto.

For the plant expression vector according to one embodiment of thepresent invention, any conventional terminator can be used. Exampleincludes, nopaline synthase (NOS), rice α-amylase RAmyl A terminator,phaseoline terminator, and a terminator for optopine gene ofAgrobacterium tumefaciens, etc., but are not limited thereto. Regardingthe necessity of terminator, it is generally known that such region canincrease a reliability and an efficiency of transcription in plantcells. Therefore, the use of terminator is highly preferable in view ofthe contexts of the present invention.

With respect to the recombinant plant expression vector according to oneembodiment of the present invention, it can be the one which isconstructed by operatively linking a gene encoding a target protein to adownstream region of the promoter of the present invention. In thepresent specification, the term “operatively-linked” is related to acomponent of an expression cassette, which functions as a unit forexpressing a heterogenous protein. For example, a promoter which isoperatively-linked to a heterogeneous DNA encoding a protein stimulatesproduction of functional mRNA which corresponds to the heterogeneousDNA.

The above-described target protein can be any kind of protein, andexamples thereof include a protein which is therapeutically useful,i.e., an enzyme, a hormone, an antibody, a cytokine, etc., a proteinwhich can accumulate a great amount of nutritional components useful forhealth enhancement in an animal including a human, and an enzyme whichcan degrade cellulose, etc., but not limited thereto. Specific examplesof a target protein include interleukin, interferon, platelet-derivedgrowth factor, hemoglobin, elastin, collagen, insulin, fibroblast growthfactor, human growth factor, human serum albumin, erythropoietin,cellobiohydrolase, endocellulase, beta-gluosidase, xylanase and thelike.

In addition, the present invention provides a method of producing atarget protein in a plant characterized in that a plant is transformedwith the above-described recombinant plant expression vector, andenvironmental stress, preferably drought stress, is applied to aresulting transgenic plant to produce a target protein in the plant. Thetarget protein which can be obtained according to this method is thesame as those described above.

Plant transformation means any method by which DNA is delivered to aplant. Such transformation method does not necessarily need a period forregeneration and/or tissue culture. Transformation of plant species isnow quite general not only for dicot plants but also for monocot plants.In principle, any transformation method can be used for introducing ahybrid DNA of the present invention to appropriate progenitor cells. Themethod can be appropriately selected from a calcium/polyethylene glycolmethod for protoplasts (Krens, F. A. et al., 1982, Nature 296, 72-74;Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), anelectroporation method for protoplasts (Shillito R. D. et al., 1985Bio/Technol. 3, 1099-1102), a microscopic injection method for plantcomponents (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), aparticle bombardment method for various plant components (DNA orRNA-coated) (Klein T. M. et al., 1987, Nature 327, 70), or a(non-complete) viral infection method in Agrobacterium tumefaciensmediated gene transfer by plant invasion or transformation of fullyripened pollen or microspore (EP 0 301 316), etc. A method preferred inthe present invention includes Agrobacterium mediated DNA transfer. Inparticular, so-called binary vector technique as disclosed in EP A 120516 and U.S. Pat. No. 4,940,838 can be preferably adopted for thepresent invention.

The “plant cell” that can be used for the plant transformation in thepresent invention can be any type of plant cell. It includes a culturedcell, a cultured tissue, a cultured organ or a whole plant. Preferably,it is a cultured cell, a cultured tissue, or a cultured organ. Morepreferably, it is a cultured cell in any form.

The term “plant tissue” can be either differentiated or undifferentiatedplant tissue, including root, stem, leaf, pollen, seed, cancerous tissueand cells having various shape that are used for culture, i.e., singlecell, protoplast, bud and callus tissue, but not limited thereto. Planttissue can be in planta or in a state of organ culture, tissue cultureor cell culture.

Further, the present invention provides a method for producing atransgenic plant comprising steps of:

transforming a plant cell with the recombinant plant expression vectorof the present invention, and

regenerating the above described transformed plant cell into atransgenic plant.

The method of the present invention comprises a step of transforming aplant cell with the recombinant vector of the present invention, andsuch transformation may be mediated by Agrobacterium tumefaciens. Inaddition, the method of the present invention comprises a step ofregenerating a transformed plant cell to a transgenic plant. A method ofregenerating a transformed plant cell to a transgenic plant can be anymethod that is well known in the pertinent art.

Further, the present invention provides a transgenic plant that isproduced by the method described in the above. The plant according tothe present invention can be food crops including rice, wheat, barley,corn, soy bean, potato, red bean, oat and millet; vegetable cropsincluding Arabidopsis thaliana, Chinese cabbage, radish, hot pepper,strawberry, tomato, watermelon, cucumber, cabbage, melon, zucchini,scallion, onion and carrot; special crops including ginseng, tobacco,cotton, sesame, sugar cane, sugar beet, wild sesame, peanut andrapeseed; fruits including apple, pear, date, peach, kiwi, grape,tangerine, orange, persimmon, plum, apricot and banana; flowersincluding rose, gladiolus, gerbera, carnation, chrysanthemum, lily, andtulip; and feed crops including rye grass, red clover, orchard grass,alfalfa, tall fescue, and perennial rye grass. Preferably, the plant canbe a monocot plant such as rice, barley, corn, wheat, rye, oat, sugarcane, onion and the like.

Further, the present invention provides a method of improving resistanceof a plant to environmental stress by introducing the promoter of thepresent invention to a plant. The environmental stress can be preferablydrought stress, but not limited thereto. This method can be carried outby operatively linking a structural gene which can improve resistance tostress to the promoter of the present invention and then introducing theresultant gene to a plant. The structural gene which can improveresistance to stress encodes a protein which is responsible forimproving resistance of a plant to environmental stress such asdehydration, low temperature, or salt stress, etc. Examples of suchprotein are as follows: LEA protein; water channel protein; a syntheticenzyme for a common solute; tobacco detoxifying enzyme; an enzyme forsynthesis of a substance which regulates osmotic pressure (e.g.,sucrose, proline or glycine betaine); a gene which encodes omega-3 fattyacid desaturase of Arabidopsis thaliana, which is an enzyme for alteringcomposition of a cell membrane and a gene which encodes D9-desaturase ofcyanobacteria; P5CS, which is a main enzyme for proline synthesis, and;AtGolS3 gene for galactinol synthesis.

Still further, the present invention provides a primer set foramplification of the promoter of the present invention, consisting of anucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3.

The primer can be an oligonucleotide which consists of a fragment of atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, and more than 29 consecutive nucleotides of SEQID NO: 1.

The primer can be an oligonucleotide which consists of a fragment of atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, and more than 28 consecutive nucleotides of SEQ ID NO: 2.

In the present specification, the term “primer” indicates asingle-stranded oligonucleotide sequence which is complementary to anucleic acid strand to be copied, and it can be a starting point forsynthesis of a primer extension product. Length and sequence of a primerare selected so as to allow the synthesis of a primer extension product.Specific length and sequence of a primer are determined in view ofprimer use condition such as temperature, ionic strength and the like,as well as complexity of a target DNA or RNA.

According to the present invention, an oligonucleotide which is used asa primer may also comprise a nucleotide analogue such asphosphorothioate, alkyl phosphorothioate or peptide nucleic acid, andalso an intercalating agent.

The present invention will now be described in greater detail withreference to the following examples. However, it is only to specificallyexemplify the present invention and in no case the scope of the presentinvention is limited by these examples.

EXAMPLES Example 1 Isolation of Genomic DNA from Whole Rice Plant

Leaves of an about three-week old rice plant (Dong-jin variety, Oryzasativa L., 1 gram) were harvested and frozen in liquid nitrogen, andthen by using a pulverizer leaf powder was prepared. With a DNeasy Plantmini kit (Qiagen), 100 g of the leaf powder was added with 400 ul AP1and 4 ul RNaseA. The mixture was kept at 65° C. for 10 minutes. Afteradding 130 ul of AP2, the mixture was kept on ice for 5 minutes followedby centrifuge at 14000 rpm for 2 minutes. Then, to removepolysaccharides, the supernatant was introduced to a spin column individed portions (650 ul each) and centrifuged. The aqueous solution wascollected and added with AP3/E which is 1.5-fold the amount of thesolution. The mixture was added to a DNA binding column in dividedportions (650 ul each) and centrifuged at 8000 rpm for 1 minute. Afterwashing twice with AW buffer (500 ul), the column was dried well andthen dissolved in sterilized water (100 ul).

Example 2 PCR Cloning of 2132 bp 996 Gene Promoter

For cloning 996 promoter, a gene-specific primer set of 996pro5′-PstI:5′-CTGCAGGGAAACGAAAAGGGGGGAAAAAAG-3′ (SEQ ID NO: 2) and 996pro3′-XbaI:5′-TCTAGACCTTCCTCTCCTTTTCCTCCTCG-3′ (SEQ ID NO: 3) was used and thegenomic DNA was amplified by polymerase chain reaction (PCR). PCRreaction solution comprised DNA 200 ng, 10×ExTaq polymerase buffersolution (pH 8.0), 200 mM dNTPs, 5× Band Doctor, 10 pmole of each primerand 5 U ExTaq polymerase (Takara Bio.). The reaction was started withoutadding any enzyme, and then amplification was carried out according tothe following condition: 1 cycle including heating at 95° C. for 5minutes as pre-denaturation step; 35 cycles including heating at 95° C.for 1 minute, 64° C. for 1 minute and 72° C. for 2 minutes, and; 1 cycleincluding heating at 72° C. for 10 minutes as a post-extension inGeneAmp PCR system 9700 amplifier (Applied Biosystem). Major productsthat had been obtained from the amplification by PCR was gel-purified byusing Gel Purification Kit (Bioneer, Korea). After subsequent cloninginto pGEM-T easy vector (Promega, USA), the plasmid constructpGEMTvec996 promoter (FIG. 1) was produced according to themanufacturer's instruction, and then sequenced (SEQ ID NO: 1).

Example 3 Construction of a Vector for 996 Promoter Expression

DNA fusion construct for the promoter which comprises a reporter geneencoding mGFP was constructed as follows. For easy cloning into a binaryvector pHC10, plasmid pGEMTvec996 promoter was digested with Pst I andXbaI, and then subcloned into Pst I and XbaI sites of pHC10. Theseclones were named pHC10::996 promoter, and an expression vector fordelivery of plant transformation was produced (FIG. 2).

Example 4 Evaluation of the Function of 996 Promoter Gene Under DroughtStress—RT-PCR Analysis

Three-week old rice leaves were dried (for 0 hr, 6 hr, 12 hr or 24 hr)and after pulverizing them with liquid nitrogen freezing, 100 mg ofthus-obtained powder was added with TRI reagent (1 ml), and left at roomtemperature for 5 minutes. Bromochloropropane (0.1 ml) was added,followed by maintaining the mixture at room temperature for 15 minutesand centrifuge at 4° C., 12000 rpm for 15 minutes. Only the supernatantwas transferred to a new tube, added with isopropanol (0.65-fold amount)and the mixture was kept at room temperature for 10 minutes. Aftercentrifuge at 4° C., 12000 rpm for 15 minutes, RNA pellet was washedwith 75% DEPC-ethanol, dried and dissolved in 30 ul solution.Gel-electrophoresis was carried out (1% agarose-formaldehyde gel) foridentification, and by using 5 ug of the total RNA, 1st cDNA wassynthesized according to the following condition: total RNA 5 ug, OligodT (10 pmole), and 10 mM dNTPs were added and the mixture was heated at65° C. for 5 minutes; 5× First strand buffer, 0.1M DTT, and RNaseOut (40U/ul) were added and the mixture was heated at 42° C. for 2 minutes and;SuperScript™ II (200 units) was added and the mixture was heated at 42°C. for 50 minutes and further at 72° C. for 15 minutes. The resultantwas used for PCR determination. By using a primer set of 996 GSP-F:5′-GGGCCTTCCTGTCTGTCGTT-3′ (SEQ ID NO: 4) and 996 GSP-R:5′-CGCACAAGGCTAAAAGCCGA-3′ (SEQ ID NO: 5), PCR amplification was carriedout according to the following condition: 1 cycle including heating at95° C. for 5 minutes as pre-denaturation step; 25 cycles includingheating at 95° C. for 30 seconds, 58° C. for 30 seconds and 72° C. for30 seconds, and; 1 cycle including heating at 72° C. for 5 minutes as apost-extension in GeneAmp PCR system 9700 amplifier (Applied Biosystem).Major products that had been amplified by PCR were confirmed bygel-electrophoresis (2.5% agarose gel; see FIG. 3).

As it is shown in FIG. 3, compared to the rice leaves which did notreceive any drought stress, gene expression was increased in the riceleaves which received drought stress. Thus, it was found that thepromoter of the present invention is a drought stress-induciblepromoter.

Example 5 GFP Determination after Transformation into Rice Callus

pHC10::996 promoter vector was introduced to the rice plant (Dong-jinvariety, Oryza sativa L.) based on Agrobacterium-mediated vacuuminfiltration method. To select a hygromycin resistant gene variant,unpolished rice in which testa was removed for callus formation was usedas rice seeds. For the removal of testa, a simple rice polishing machinewas used and the rice seeds with removed testa were sterilized once with70% ethanol for 10 minutes, followed by 30 minutes sterilization in 2 to3% sodium hypochlorite solution and washing with sterilized water atleast three times. The rice seeds obtained after the sterilization wereplaced on a medium for callus formation (i.e., 10 to 12 seeds permedium) and incubated under dark condition at 28? for 3 to 4 weeks.Then, light yellow callus (1 to 2 mm diameter) was selected and culturedin 2N6 medium for three days. Meanwhile, the day after the callusselection, Agrobacterium was added to a medium comprising carbenicillinand hygromycin and cultured under dark condition at 28° C. for 3 to 5days. After transferring the callus which had been cultured for threedays, it was co-cultured in suspension comprising Agrobacterium andfurther cultured under dark condition at 25? for 3 days. The resultingco-cultured callus was washed with sterilized water comprisingcarbenicillin and the transformed callus was cultured under darkcondition at 28° C. for 2 to 3 weeks. Healthy callus showing good growthwas selected again. After the transfer to a new medium, the callus wasagain cultured under the same condition. Healthy callus was then againadded to a regeneration selection medium. The transformant in which thetarget gene had been introduced was identified by genomic PCR.

GFP Determination

In order to examine the function of 996 promoter, an ELISA analysisusing an GFP antibody was carried out. Specifically, after applying 0 hror 6 hr drought stress to the transformant leaf which had been selectedby genomic PCR, total protein was extracted by using a solutioncomprising 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 8 mM MgCl2, and aproteinase inhibitor. Thus-obtained total protein was quantified byusing Bradford assay solution, and 10 ug of the protein was used for thedetermination. In a microplate, the antigen comprising 200 ul coatingbuffer and the total protein was added and reacted at 4° C. for 12hours. After blocking with 1×PBS comprising 4% skim milk for two hoursat room temperature, GFP antibody was diluted to 1/4000 with 1×PBScomprising 1% skim milk and was allowed to bind to the antigen byincubating them at room temperature for 2 hours. 1×PBS comprising 0.05%Tween 20 was used for washing, and the secondary antibody was diluted to1/2000 with 1×PBS comprising 1% skim milk and was allowed to bind to theantigen by incubating them at room temperature for 1 hour followed bywashing. After the development with a solution comprising 30% H2O2, PCbuffer and OPD, the reaction was terminated by adding 2.5M H2SO4. Then,the absorbance at 490 nm was determined by using a microplate reader andthe result was analyzed (FIG. 4).

As it is shown in FIG. 4, compared to the transgenic rice which did notreceive any drought stress, GFP expression was significantly increasedin the transgenic rice which received drought stress. Thus, it was foundthat the promoter of the present invention is a drought stress-induciblepromoter.

1. Environmental stress-inducible 996 promoter isolated from rice whichconsists of nucleotide sequence of SEQ ID NO:
 1. 2. The promoteraccording to claim 1, characterized in that the environmental stress isdrought stress.
 3. A recombinant plant expression vector comprising thepromoter of claim
 1. 4. The recombinant plant expression vectoraccording to claim 3, which is constructed by operatively linking atarget gene encoding a target protein to a downstream region of thepromoter.
 5. A method of producing a target protein in a plant,characterized in that a plant is transformed with the recombinant plantexpression vector of claim 4 and drought stress is applied to aresulting transgenic plant to produce the target protein in the plant.6. The method according to claim 5, characterized in that the targetprotein is selected from a group consisting of interleukin, interferon,platelet-derived growth factor, hemoglobin, elastin, collagen, insulin,fibroblast growth factor, human growth factor, human serum albumin,erythropoietin, cellobiohydrolase, endocellulase, beta-gluosidase, andxylanase.
 7. A method for producing a transgenic plant comprising stepsof: transforming a plant cell with the recombinant plant expressionvector of claim 3, and regenerating the above described transformedplant cell into a transgenic plant.
 8. A transgenic plant which isproduced according to the method of claim
 7. 9. The transgenic plantaccording to claim 8, characterized in that said plant is a monocotplant.
 10. A method of improving resistance of a plant to environmentalstress by introducing the promoter of claim 1 to a plant.
 11. The methodaccording to claim 10, characterized in that the environmental stress isdrought stress.
 12. A primer set for amplification of the promoter ofclaim 1, consisting of nucleotide sequence of SEQ ID NO: 2 and SEQ IDNO: 3.